The present invention relates to a method for efficiently producing a large amount of plantlets by means of tissue culture using differentiation totipotency of a plant.
When dedifferentiated cells obtained by tissue culture of a plant are cultured under appropriate conditions, redifferentiation of organs can be induced to regenerate a complete plant. Up to now, numerous studies have already been made on a method of producing a redifferentiated plant such as a plantlet by using a tissue culture technique. Examples of the studies include xe2x80x9cdevelopment of medium suitable for growth promotionxe2x80x9d (Jpn. Pat. Appln. Publication No. 7-196410), xe2x80x9cstudy on culture conditionsxe2x80x9d (Jpn. Pat. Appln. Publication No. 9-220036), xe2x80x9cutilization of substance released from plantxe2x80x9d (Jpn. Pat. Appln. Publication No. 6-327368), and xe2x80x9cdevelopment of culture mediumxe2x80x9d (Jpn. Pat. Appln. Publication No. 8-140513). However, the studies on medium and culture conditions must be separately performed for each plant species. Therefore, these approaches have a drawback since its general applicability is low. Then, to obtain the redifferentiated plant such as a plantlet in a large amount, it is desired to develop a widely applicable, efficient, and simple method of producing the redifferentiated plantlet by using the tissue culture technique.
An object of the present invention is to provide a method of producing a redifferentiated plant such as an adventive embryo and plantlet, efficiently in a large amount in a short period.
To attain the aforementioned object, in the present invention, a thickening agent was added to a growth medium for cultured plant cells. As a result, it was found that the number of redifferentiated plantlets was increased. It has never been reported that productivity of the redifferentiated plantlets is improved by addition of the thickening agent, as is the case of the present invention.
The method of producing a redifferentiated plantlet of the present invention comprises a step of proliferating cultured plant cells in a liquid medium containing a thickening agent and a step of redifferentiating the cultured plant cells after the proliferating step.
Now, the efficient production method for a redifferentiated plantlet of the present invention will be explained in detail.
The method of the present invention comprises a first step of proliferating cultured plant cells in a liquid medium containing a thickening agent and a second step of redifferentiating the cultured plant cells after the proliferating step. In this case, the culture of the first step is called xe2x80x9cgrowth culturexe2x80x9d and the culture of the second step is called xe2x80x9credifferentiation culturexe2x80x9d.
The type of plant cell to be used in the efficient production method for a redifferentiated plantlet of the present invention is not particularly limited. Any type of cell may be used as long as it can be redifferentiated into an individual plant. Furthermore, the type of cell may be derived from any species and may be a cultured cell induced from any site of a plant. The cultured cells for producing the redifferentiated plantlet may be subcultured cells in a state of callus, or any cells already cultured in a liquid culture medium. Alternatively, protoplasts may be used. Of them, it is preferable to use the cultured cells maintained in a liquid medium such as cultured carrot cells. Note that induction of the callus, preparation of the liquid-cultured cells, and preparation of the protoplasts can be performed by a conventionally-known method to those skilled in the art, more preferably, by appropriately modifying it if necessary.
The liquid medium for use in culturing and proliferating cultured cells is not particularly limited, and any culture medium may be used as long as the cultured cells can be proliferated. In general, preferably used is an aqueous medium containing Murashige-Skoog medium (Murashige T. and Skoog. F (1962) Physiol. Plant. 15, 473) as a main component and a requisite sugar and plant hormones, etc. as additives. As the sugar, for example, sucrose is added to a basic medium such as the Murashige-Skoog medium in an amount of, e.g., 10-50 g/L. As the plant hormones, auxins and cytokinins are used. As the auxins, 2,4-dichlorophenoxy acetic acid may be used in an amount of 0.05-5 mg/L. As the cytokinins, kinetin may be used in an amount of 0.05 to 0.5 mg/L. The medium to be used preferably has pH 5.0-6.5.
In the present invention, the thickening agent contained in the medium is not particularly limited, and any thickening agent may be used as long as it increases a viscosity of the medium and has no adverse effect upon growth of the cultured cells. Examples of the thickening agent include sodium alginate, propylene glycol alginate ester, carboxymethyl cellulose, methyl cellulose, carboxymethyl starch, sodium polyacrylate, guar gum, xanthan gum, and locust bean gum. Preferably, sodium alginate and carboxymethyl cellulose may be used. The thickening agent may be added by dissolving it in a medium when the medium is prepared.
The concentration of the thickening agent in the medium can be appropriately set within a range from a concentration imparting a viscosity to the medium to a concentration imparting a viscosity with no effect upon growth of the cultured cells. For example, sodium alginate or carboxymethyl cellulose may be contained in an amount of 0.05-0.5 (w/v) %, and preferably in an amount of 0.1-0.2 (w/v) %.
In the present invention, the step of culturing/ proliferating the cultured cells in a liquid medium containing the thickening agent can be aseptically carried out in accordance with a known method in the art. For example, cells are cultured in an MS medium containing plant hormones and a sugar while gyrating or shaking. This growth culture is performed preferably at a gyration speed (or shaking speed) of 50-200 rpm (or 50-200/min) at a temperature of 20-30xc2x0 C. under dark conditions.
In the present invention, the growth culture is performed for 10 to 20 days. Thereafter, the cultured cells are transferred to a redifferentiation culturing step.
In the present invention, the step of redifferentiating the cells proliferated in the growth medium in accordance with the aforementioned method, can be carried out by appropriately modifying a known method to those skilled in the art depending upon a type of cultured cells. Generally, to induce redifferentiation, the cultured cells are inoculated to a medium (redifferentiation medium), whose composition of plant hormones are different from that in the growth medium and suitable for inducing redifferentiation.
The medium (redifferentiation medium) with the composition of the plant hormones suitable for redifferentiation must be studied and selected depending upon a type of the cultured cells. For example, as a redifferentiation medium for cultured carrot cells, it is possible to use a medium which is the same as the growth medium mentioned above except that plant hormones is eliminated, as will be described later. When the cultured carrot cells are inoculated to the redifferentiation medium excluding the plant hormones and then cultured in the medium, the cultured cells are regenerated into organs (stem, leaf, and root) via an adventive embryo.
Besides the aforementioned method for producing a redifferentiated plantlet by eliminating the plant hormones in the growth medium, in another plant species, another method may be used. In this method, redifferentiation of organs such as stem, leaf, and root may be sequentially induced by using auxins and cytokinins in combination and by changing the quantity ratio thereof.
In a case where redifferentiation is induced, the culture conditions must be also studied as to whether or not they have preferable effects upon the redifferentiation with respect to individual plant species and selected based upon the results of the studies.
For example, in carrot cultured cells, the redifferentiation culture step is carried out under light conditions as will be described later.
According to the production method of the present invention, the cultured cells are first grown in the medium containing a thickening agent for a period. Then, the resultant cultured cells are transferred to the redifferentiation culturing step. Thereafter, the number of redifferentiated plantlets is counted. The counted number is compared with that of a control grown in a medium containing no thickening agent. In this manner, effect of the present invention can be demonstrated.